In lambs, weaning imposes stress that can contribute to impaired rumen epithelial barrier functionality and immunological dysregulation. In this study, the effects of a yeast co-culture consisting of Saccharomyces cerevisiae and Kluyveromyces marxianus (NM) on rumen health in lambs was evaluated, with a focus on parameters including growth performance, ruminal fermentation, and epithelial barrier integrity, ruminal metabolic function, and the composition of the ruminal bacteria. In total, 24 lambs were grouped into four groups of six lambs including a control (C) group fed a basal diet, and N, M, and NM groups in which lambs were fed the basal diet respectively supplemented with S. cerevisiae yeast cultures (30 g/d per head), K. marxianus yeast cultures (30 g/d per head), and co-cultures of both yeasts (30 g/d per head), the experiment lasted for 42 d. Subsequent analyses revealed that relative to the C group, the average daily gain (ADG) of lambs in the NM group was significantly greater and exhibited significant increases in a range of mRNA relative expression including monocarboxylate transporter 1 (MCT1), (Na⁺)/hydrogen (H⁺) exchanger 1 (NHE1), (Na⁺)/hydrogen (H⁺) exchanger 3 (NHE3), proton-coupled amino acid transporter 1 (PAT1), vacuolar H⁺-ATPase (vH+ ATPase), claudin-1, occludin in the rumen epithelium (P < 0.05). Compared with the C group, the pH of the rumen contents in the NM group was significantly decreased , and the concentrations of acetate, propionate, and butyrate were significantly increased (P < 0.05). Analysis of the rumen bacteria showed that the NM group exhibited increases in the relative abundance of Prevotella, Treponema, Moryella, Fibrobacter, CF231 and Ruminococcus (P < 0.05). Metabolomics analyses revealed an increase in the relative content of phthalic acid and cinnamaldehyde in the NM group as compared to the C group (P < 0.05), together with the greater relative content of L-tyrosine, L-dopa, rosmarinic acid, and tyrosol generated by the tyrosine metabolic pathway (P < 0.05). Spearman's correlation analyses revealed relative abundance levels of Fibrobacter and Ruminococcus were positively correlated with the mRNA relative expression levels of PAT1, NHE3, and zonula occluden-1 (ZO-1), as well as with tyrosol, phthalic acid, and cinnamaldehyde levels (P < 0.05). Ultimately, these results suggest that dietary supplementation with NM has a wide range of beneficial effects on weaned lambs and is superior to single bacterial fermentation. These effects include improvements in daily gain and rumen epithelial barrier integrity, as well as improvements in the composition of the rumen microbiome, and alterations in tyrosine metabolic pathways.

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